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Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785- 1790.

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Page 1: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate

Zhang Hui

2012213034

J. Agric. Food Chem.2002,50,1785-1790.

Page 2: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Contents

Introduction

Materials and methods

Results and discussion

Conclusion

Page 3: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Introduction

Tannins

hydrolyzable tannins

condensed tannins high molecular weight polyphenolics found in

higher plants

gallotannins

ellagitannins

HO

HO

HO

COOH HO

HO

OH

OH

O

O

O

O

Page 4: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Structural formulas of representative hydrolyzable tannins from plants. Compound 1 is pentagalloyl glucose, comprised of the gallate (G,2) esters of glucose. Compound 3 is the simple ellagitannin eugeniin, comprised of both the gallate esters and a single HHDP (G−G,4) ester. Compound 5 is EGCG, the gallate ester of the flavanoid epigallocatechin. Compound 6 is oenothein B, a macrocyclic hydrolyzable tannin. All four of thehydrolyzable tannins react in acidic methanol to yield methyl gallate (7)

OOO

OO

O

GG

G

G

G

1

G=OH

OH

OH

O

2

OOO

OO

O

GG

G

G

G

3

O

OH

OH

OHHO

OH

OO

OH

OHHO

5

OH

OH

OH

O

O

7

OHHO

HO

O

HO OH

OH

OG-G=

4

HO

HO

OH

O

O

HO

HO

O

O

O

O

OO

OH

OH

HO

HO

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

O

G

O

OO

OH

OO

O

G

6

Page 5: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Introduction

Individual hydrolyzable tannins can usually be determined by HPLC, but methods for conveniently screening large numbers of samples with variable hydrolyzable tannin compositions have not been developed.

The KIO3 method has been employed in some studies of oaks and maples.

The purpose of this work was to optimize the KIO3

method for analysis of hydrolyzable tannins.

Page 6: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Materials and methods Reagents tannin acid , gallic acid, methyl gallate, epigallocatechin

gallate (EGCG) Methanolysis and pH Adjustment anhydrous methanol, 85 , 20h℃ , pH: 5.5±0.1 Analysis 30 ℃ water bath, 50 min after KIO3 was added, recorded absorbance at 525 nm HPLC Analysis of Methanolysis Products C18 (100×4.6 mm),1.0 mL/min , ƛmax 525 nm, gradient elution

Page 7: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Results and Discussion

OH

OH

OH

O

+ KIO3 A B

max 525 nmƛ yellow

21

Why KIO3 ?Hypochlorite ? the lifetime of A was shorterPotassium periodate (KIO4) ?past the red intermediate to the final product

Page 8: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Disadvantages of KIO3 method

different color yields

in rates of reaction

for structurallydifferent gallotannins

Improve

Why not gallic acid ?

Methyl gallate

OH

OH

OH

O

O

H3C

+ KIO3 A B

max 525 nmƛ yellow

21

Page 9: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Why anhydrous methanol?

traces of water caused production of a mixture of gallic acid and methyl gallate

avoid the disappearance of some methyl gallate formation of unidentified phenolic products observed at higher temperatures

Why 85℃?

Methanolysis

Page 10: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Reaction of KIO3 with methyl gallate as a function of pH. Methyl gallate solutions dissolved in methanol were diluted with a series of solutions comprised of ethanolamine and ammonium acetate buffer prepared at various pH values between 4.5 and 7.0.

How to control pH?

too basic, oxidation was acceleratedand only the yellow end product

too acidic, the chromophore Adid not accumulate

KIO3 Reaction

Page 11: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

50 min after KIO3 was added, recorded absorbance at 525 nm

How to control time?

product A was temperature-dependent and time-dependent

at room temperature, there were significant day-to-day variations

How to control temperature?

30 ℃ water bath

KIO3 Reaction

Page 12: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

a linear relationship between methyl gallate and absorbance at 525 nm:

Abs = 0.0132 μg + 0.0701

where R2 = 0.996, standard error of Y = 0.0303, and standard error of slope = 0.000 19.

Page 13: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Conversion of hydrolyzable tannins to methyl gallate by 20 h of methanolysis at 85°C.

A Methanolysis samples (s) of tannic acid

B . EGCG

C. Acer leaf powder

Page 14: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Theoretical Production of Methyl Gallate from Hydrolyzable Tannins

Yield of methyl gallate is calculated from molecular weights of tannin andmethyl gallate and is expressed in grams of methyl gallate per gram of tannin.

HO

HO

OH

O

O

HO

HO

O

O

O

O

OO

OH

OH

HO

HO

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

O

G

O

OO

OH

OO

O

G

Page 15: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Analysis was done with the original KIO3 method (A)

Analysis was done with the new method described (B)

Example:red maple leaves

Page 16: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Theoretical Production of Methyl Gallate from Hydrolyzable Tannins

Yield of methyl gallate is calculated from molecular weights of tannin andmethyl gallate and is expressed in grams of methyl gallate per gram of tannin.

Page 17: Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790

Conclusion

The new KIO3 method will be useful for

examining hydrolyzable tannin in ecological,

physiological, and nutritional studies of plants.